Main findings
The main findings of this study were that the 5-HT3A receptor is highly expressed in human masseter and tibialis muscle tissue, but that there were no differences in expression due to sex. Further, there were more immunoreactive nerve fibers expressing 5-HT3 receptors and NaV1.8 sodium channels in the masseter muscle of female patients with myofascial TMD compared to healthy female controls. These findings indicate that 5-HT3 receptors might be up-regulated in myofascial TMD and may therefore play a role in pain transmission, hence providing the 5-HT3-receptor as a target for future therapeutics or as a potential biomarker of chronic muscle pain.
Secondarily, the results from this study indicate that the microbiopsy technique provided sufficient muscle tissue, less discomfort and post-surgical pain than the traditional biopsy technique. Therefore, the microbiopsy technique can be regarded as a valid biopsy method for immunohistochemical analyses of muscle tissue, and may, thus, be preferred to traditional open biopsies in future studies or even as a diagnostic tool.
Receptor expression
The immunohistochemical analysis of the muscle sections indicates that the 5-HT
3A receptor is highly expressed by human masseter and tibialis anterior muscle nerve fibers. To our knowledge there are no other studies that have studied the expression of the 5-HT3 receptor in human muscle tissues. A recent study did show that the NMDA receptor is also expressed by masseter muscle nerve fibers (45-50%) in humans and rats [
22]. However, results from animal studies regarding the expression of the 5-HT
3 receptor in trigeminal and dorsal root ganglion neurons [
7,
38] are consistent with the present study. It is well established that the Na
V1.8 sodium channels play an important role in nociception and chronic pain when found on small unmyelinated and thinly myelinated sensory neurons [
24,
25,
29,
30,
39] since they contribute to action potentials in sensory neurons [
28,
39,
40] and are involved in peripheral sensitization [
23]. They have further also been found in large diameter sensory neurons in human pain states [
41], as well as large diameter fibers in mouse [
42] and rat [
26,
43]muscle afferent neurons. Hence, this TTX resistant sodium channel is not just limited to small diameter nerve fibers [
44]. Inflammation has been shown to enhance nociceptive signaling by the Na
V1.8 sodium channels [
31,
44‐
46], in part, through release of inflammatory mediators, such as 5-HT and prostaglandin E
2, which directly increase Na
V1.8 mediated currents [
31,
47]. Also, 5-HT is known to participate in pain mediation at the peripheral level, acting on the 5-HT
3 receptors, which mainly appear on afferent nociceptive sensory, autonomic and enteric neurons [
16]. Given this, one can assume that the Na
V1.8 positive fibers are sensory neurons with a potential nociceptive function, consequently implying that the muscle nerve fibers that co-express the 5-HT
3A receptors and Na
V1.8 sodium channels are most probably nociceptors.
Noteworthy is the significantly higher average of fibers per section in the masseter muscle of patients with myofascial TMD, which could indicate a proliferation of nerve fibers in the painful muscle tissue. There is some evidence that hyper-innervation of the tendon with putative nociceptive fibers occurs in Achilles tendinosis [
48,
49]. The larger number of fibers in tender regions of the masseter muscle of TMD patients found in this study could indicate an up-regulation of 5-HT
3 receptors. This finding may explain previous findings that a local injection of the 5-HT
3 receptor antagonist granisetron was effective in decreasing muscle sensitivity in patients with localized myalgia [
21] and fibromyalgia [
50].
The majority of the 5-HT
3A and Na
V1.8 positive fibers in the masseter muscle were found in the connective tissue, which suggests that masticatory muscle pain may emanate primarily from the connective tissue. This theory is based on the findings from the muscle sections where both the patients and healthy subjects had significantly more Na
V1.8 positive nerve fibers in connective tissue compared to fibers associated with myocytes. The elevated levels of serotonin in the masseter muscle in patients with myofascial TMD [
3] and the very high expression of 5-HT
3A and Na
V1.8 positive fibers on sensory nerves associated with connective tissue, shown in this study, support the theory that the muscle nociceptors in painful muscles could be sensitized by serotonin and hence that serotonin may have an important role in myofascial TMD.
Apart from pain mediation, the 5-HT
3 receptors are involved in many events in the human body. Centrally they play an important role in psychosis, anxiety, cognition and eating disorders [
51], but also in motor system function. A recent study showed that activation of the 5-HT
3 receptor induced rhythmic movements of the hindlimbs in mice [
52]. In the periphery they have a well-known role in emetic pathways [
53] and they have a role in the intestinal tone, activating colonic migrating motor complex, i.e. muscular motor activity [
54]. PGP 9.5 labels both small and large diameter myelinated fibers [
55] and Na
V1.8 is a marker of small diameter thinly myelinated nociceptive fibers [
44]. With this in mind, as the majority of the 5-HT
3A positive muscle nerve fibers were located near myocytes and did not co-express Na
V1.8, our findings imply that the 5-HT
3A positive fibers in association with the myocytes are likely either proprioceptive muscle spindle afferent fibers or motor axons [
56].
Comparison of the biopsy techniques
The results of this study indicate that the microbiopsy technique provided sufficient muscle tissue for immunohistochemistry and led to less discomfort and post-surgical pain as well as fewer visits than the traditional biopsy technique. This is in agreement with a previous study comparing the microbiopsy technique with the traditional open biopsy technique in the vastus lateralis of the quadriceps femoris muscle [
57]. Further, the microbiopsy technique seems to be more precise, since by using the Bard®TruGuide™ coaxial needle, the surgeon is more likely to remove tissue from the exact region of interest without damaging the surrounding tissue [
35]. It was also shown that the muscle sections from the microbiopsies contained less of the surrounding tissues, such as parts of the salivary glands than traditional open biopsies (Table
4). Taken together this shows that the microbiopsy technique is a valid biopsy method for immunohistochemical analyses of muscle tissue and hence, may be preferred to traditional open biopsies in future studies or even as a diagnostic tool.
Differences due to sex or anatomical localization
In contrast to a previous study [
7] indicating that the expression of 5-HT
3 receptors by trigeminal ganglion neurons innervating the masseter muscle was higher in female rats, this study showed no significant difference in the expression of 5-HT
3A receptors in the masseter or tibialis muscles, when men and women were compared.
In previous studies, women reported more pain than men when serotonin was injected into the masseter muscle [
13], and a larger pain area when hypertonic saline was injected [
10]. Based on those studies one could have expected a significant sex difference with a larger amount of nerve fibers co-expressing 5-HT
3A receptors and Na
V1.8 sodium channels in women. Further, a previous human experimental study showed that systemic administration of the 5-HT
3 receptor antagonist granisetron increased the PPT over the tibialis anterior muscle in men but not in women [
11]. As a possible explanation for this difference could have been that the number of receptors differs between men and women, however, the findings of the present study do not support this explanation. It should be noted that a limitation of this study is the small sample size (6–7 individuals of each sex), which may have been insufficient to permit identification of sex-related differences in 5-HT
3A receptor expression.
There was a difference between the two skeletal muscles since a majority of the fibers co-expressing 5-HT
3A receptors and Na
V1.8 sodium channels were found in the connective tissue in the masseter muscle while in the tibialis muscle these fibers were found to be associated with myocytes. The tibialis muscle is rarely exposed to chronic pain in contrast to the masseter muscle and is further innervated by the deep peroneal nerve which major role is to contract the muscle fibers in order to dorsiflex and invert the foot [
58]. In contrast to the tibialis anterior muscle, the masseter muscle expressed a larger amount of putatively nociceptive nerve fibers in the connective tissue. This is an interesting difference between muscles that could be a consequence of the different innervation with either trigeminal branches (masseter muscle) or spinal branches (tibialis muscle) and functionality of the nerves regarding motor and sensory functions [
58]. Therefore, the larger amount of putatively nociceptive nerve fibers in the connective tissue of the masseter muscle and the difference in innervation might partly explain the more frequent presence of chronic myalgia in the masseter muscle.
Study limitations
One limitation of the study is, as mentioned, the small sample size which might have affected the outcome regarding sex-related differences. Another possible limitation of the study is the exclusion criterion “use of analgesic or anti-inflammatory medication during the 24 hours preceding the biopsy”. This criterion was primarily in order to avoid any risk of including a patient with myofascial TMD among the healthy participants. However, 24 hours is a short wash-out period for NSAIDs which could have affected the biopsy procedure negatively, for example through increased bleeding, since the use of NSAIDs might impair the thrombocyte aggregation for up to 8–10 days. Use of NSAID might also have suppressed receptor expression. If so, it is likely that patients with myfascial TMD would have been affected more and in that case the difference in frequency would be even greater than found in the current study. The storage of the biopsies in paraffin could be another possible limitation since this technique might blunt the antigenicity of the samples, which may make it more difficult to detect the receptors. In future studies the muscle biopsies are suggested to be frozen immediately in -80°C.